The birth, death, and evolutionary compensation of uORFs in Drosophila

Upstream open reading frames (uORFs) play crucial roles in regulating mRNA translation; however, the evolutionary dynamics of uORFs and the potential coevolution of uORF gain and loss remain largely unexplored. Here, we comprehensively analyze the evolutionary landscape of uORFs across Drosophila species, revealing a complex pattern of birth and death events throughout the phylogeny, with a net gain trend dominating the overall pattern. We identified instances of evolutionary compensation in which the loss of a uORF was accompanied by the gain of another within the same gene, or vice versa. Surprisingly, a significant proportion of fixed uORF loss events in D. melanogaster were driven by positive selection. Dual-luciferase assays revealed that approximately 75% of the tested uORF gains or losses altered translational regulation, with some exhibiting condition-specific effects. Surveying uORF-related variants across 1,356 D. melanogaster genomes revealed strong purifying selection, with some variants potentially contributing to local adaptation. Our findings highlight the crucial role of natural selection in shaping the evolutionary landscape of uORFs in Drosophila and provide novel insights into the complex interplay between the evolution of coding and regulatory elements.